The Novel Function And Target Of Con-T[M8Q] On Morphine Analgesia Tolerance In Mice

N-methyl-D-aspartate receptor(N-methyl-D-aspartate receptor) is one of the glutamate receptors and is closely related to the formation and maintenance of synaptic plasticity, epilepsy and drug addiction, etc. NMDA receptors are comprised of multiple subtypes, such as NR1 and NR2(A-D), and the 2B subunit(N R2B) plays an important role in a variety of neuro pathic pain, inflammatory pain, drug addiction and other pathological processes. Conantokins, a kind of natural conus peptides, can specifically act on NMDA receptors. Our previous studies show that conantokins exhibit analgesic activity and those with higher selectivity at the NR2 B subunit possess higher analgesic activity, and find that con-G and its mutants can significantly suppress the naloxone-precipitated withdrawal jumps in the morphine- induced mice.Based on the above previous research, this subject will investigate the new functions of inhibition of con-T[M8Q] on morphine analgesic tolerance. In addition, based on the structure- function relationship and targets of con-T and con-G as well as molecular calculation, we want to determine the binding sites of con-T[M8Q] at NMDA NR2 B using the patch-clamp technique. This work will be of great importance in developing new analgesic peptides, narcotic ana lgesic agents and studying the mechanism of interaction between conantokin peptides and NMDA receptors. In addition, we will synthesize several new conotoxins. The main results are as follows:1. The hot plate test and acetic acid writhing test were used to determine the effect of low dose of con-T[M8Q](5 nmol/kg, 10 nmol/ kg, 20 nmol/kg, i.c.v.) on the expression of morphine tolerance. The results indicated that con-T[M8Q] significantly increased the PMAP and reduced the writhing times in morphine tolerance mice. 2. The hot plate test and acetic acid writhing test were also used to determine the effect of con-T[M8Q](5 nmol/kg, 10 nmol/ kg, 20 nmol/kg, i.c.v.) on the development of morphine tolerance in mice. The results showed that con- T[M8Q] significantly inhibited the development of morphine tolerance in mice, and its inhibitory activity was significantly high than Ifenprodil, a small molecular antagonist of the NMDA receptor NR2 B subtype. 3. The analgesic activity and addiction of con-T[M8Q] were determined. The results showed that con- T[M8Q] exhibited a low analgesic activity and did not displaymorphine- like addiction, suggesting that the inhibition of con-T[M8Q] for morphine tolerance in mice derived from its inhibitory activity on NMDA, but not from its analgesic activity. 4. According to the structure-activity relationship of con-T(Y5,Q8 and K19 are functional amino acids) and the molecular calculation of interaction between conantokins and NMDA receptor N R2 B subunit performed by a professor from Huazhong University of Science and Technology, we designed some variants of NMDA receptor NR2 B subunit. Up till now, we have constructed the NR2 B subunit and successfully expressed it in HEK293 cells, and have done the binding experiment of con-T[M8Q] for NMDA receptor NR2 B subunit. 5. We have previously cloned a variety of new gene sequences of conus peptides. In order to determine their functions, we synthetized two conopeptides-Bt3.1 and Im IIA.